Practical Synthesis and Application of Halogen-Doped Pyrrole Building Blocks

A practical access to four new halogen-substituted pyrrole building blocks was realized in two to five synthetic steps from commercially available starting materials. The target compounds were prepared on a 50 mg to 1 g scale, and their conversion to nanomolar inhibitors of bacterial DNA gyrase B was demonstrated for three of the prepared building blocks to showcase the usefulness of such chemical motifs in medicinal chemistry.


INTRODUCTION
Halogen-substituted pyrrole-2-carboxamide is an integral molecular fragment of bioactive marine natural products as well as natural and synthetic anti-infectives ( Figure 1). In particular, mono-and dibromopyrrole-2-carboxamide are found in oroidin 1 and hymenidin, 2 which are postulated precursors for structurally diverse mono-and oligomeric secondary metabolites involved in the chemical defense of Agelas marine sponges. A representative compound ageliferin 3 features a complex multichiral scaffold. 4 Furthermore, 3,4dichloro-5-methyl-1H-pyrrole-2-carboxamide is a molecular fragment of natural 5,6 and synthetic 7 antibacterials, crucial for binding to the active site of bacterial topoisomerases, and the 3-fluoro-1H-pyrrole-2-carboxamide moiety is found in promising preclinical candidates, active against hepatitis B virus ( Figure 1). 8 During our ongoing research in the field of dual bacterial DNA gyrase/topoisomerase IV inhibitors, 9−15 a promising hit compound 1 (Figure 2) was identified, displaying low nanomolar inhibition of the target enzymes and broadspectrum activity against gram-positive bacterial strains. 16 Due to the compound's high lipophilicity, its more polar analogues 2−5 were designed by varying the 3,4-dichloro-5methyl-1H-pyrrole moiety, envisioning improved physical properties (c log P was calculated by ChemDraw) of the analogues while retaining the on-target activity ( Figure 2).
With no preceding literature on the synthesis of the required pyrrole building blocks for the preparation of compounds 2−5, we report herein our synthetic endeavors, where the main goal  was the timely delivery of at least 100 mg of the sample to be built into the bioactive molecules. The amide bond of the target compounds can be formed using pyrrole-2-carbonyl chloride or 2-trichloroacetylpyrrole; therefore, either would be an acceptable option.

RESULTS AND DISCUSSION
The literature procedure for the synthesis of 4-chloro-5methyl-1H-pyrrole-2-carboxylic acid involves chlorination of ethyl 5-methyl-1H-pyrrole-2-carboxylate using N-chlorosuccinimide at 0°C and required in our hands laborious chromatographic separation of two barely resolved products. 17 The practical synthesis of an alternative acylating agent 8 for the introduction of the same structural fragment was thus developed (Scheme 1). Trichloroacetylpyrrole 7 was prepared from pyrrole-2-carbaldehyde 6 employing the Wolff−Kishner reduction and Friedel−Crafts acylation. 18 It was then directly monochlorinated using N-chlorosuccinimide at r.t. and the pure product 8 was obtained on the gram scale in 61% isolated yield after convenient crystallization from dichloromethane. Its structure was unambiguously determined by two-dimensional (2D) nuclear magnetic resonance (NMR) experiments (Supporting Information), showing that the electrophilic chlorination was selective for the position next to the electron-donating methyl substituent.
Next, we targeted the 4-fluoro-substituted building block. The screening of different halogen exchange ("Halex") conditions involving crown ether 18-C-6 and [2.2.2]cryptand, for the conversion of chloropyrrole 8 or ethyl 4-chloro-5methylpyrrole-2-carboxylate to the corresponding arylfluorides, returned no hits. 19 We thus resorted to electrophilic fluorination of ethyl 5-methyl-1H-pyrrole-2-carboxylate 9 (Scheme 2). Initial 0.5 mmol scale screening of the reaction conditions (Table S1 in the Supporting Information) revealed that Selectfluor-mediated fluorination 20 outperformed the Nfluorobenzenesulfonimide (NFSI)-mediated Lewis acid-catalyzed fluorination, 21 as the former resulted in somewhat cleaner conversions. When the fluorination was performed at 0°C in a mixture of acetonitrile and acetic acid (Table S2, entries 11 and 12), the formation of target compound 10, accompanied by an acetoxy side product 11, was observed. Their structures were confirmed by single-crystal X-ray diffraction analysis (Figures S1 and S2 in the Supporting Information). Aiming for an efficient med−chem synthetic route, the reaction was performed on a 2 g scale (Scheme 2), delivering 10 in a consistent 4.5−6.5% yield after flash chromatography. Ester 10 was hydrolyzed to acid 12, requiring rather forcing conditions, and acyl chloride 13 was finally formed using oxalyl chloride in dichloromethane. It is noteworthy that acyl chloride formation using refluxing sulfonyl chloride or oxalyl chloride with the catalytic quantity of dimethylformamide (DMF) resulted in the formation of a significant amount of unidentified side products.
Ethyl 3-fluoro-1H-pyrrole-2-carboxylate 14 has recently become commercially available at a reasonable price because it is a key building block for a drug candidate against hepatitis B virus. 22 This was a good starting point for the synthesis of 3fluoro-5-methyl-1H-pyrrole-2-carboxylic acid 18 (Scheme 3).
The Vilsmeier−Haack formylation of 14 gave at 68% conversion a 43:57 mixture of 4-and 5-formylated regioisomers 16 and 15, which were separated by flash chromatography. The regioisomers' identity was assigned by 19 F NMR as follows: 4-formyl isomer 16 features a singlet and 5-formyl isomer 15 features a doublet, 3 J F,H = 4 Hz, confirming the presence of a vicinal proton. Moreover, the 13 C NMR peak of the formyl carbon of 15 is a singlet and that of 16 is a doublet, 3 J C,F = 2.8 Hz.
Based on the literature reports on the reduction of estercontaining formylpyrroles to methylpyrroles, 23 we first attempted a BH 3 ·THF-mediated reduction of 15−17, which in this case yielded the intermediate alcohol; no full reduction was observed even after several days of stirring with periodical addition of excess BH 3 ·THF. Other literature reports on aldehyde-to-methyl reduction in the presence of ester include a two-step Mozingo protocol via thioketal. 24 Aiming to secure a   convenient one-pot procedure, we opted for the modified C l e m m e n s e n r e d u c t i o n . 2 5 A dioxane-soluble [ZnCl 2 (dioxane) 2 ] complex 26 was prepared by treating zinc dust with anhydrous 4 M HCl in dioxane. This proved to be a very efficient and reasonably selective reduction medium, delivering 17 after 40 min at r.t. in 20% isolated yield. Optimization of the reaction conditions and elucidation of the mechanism is beyond the aim of this study; however, we speculate that a dioxane-soluble Zn(II) species forms a zinc− ylide intermediate more efficiently compared to the classical heterogeneous Clemmensen reduction (Zn/Hg/HCl/H 2 O), allowing the reaction to proceed at room temperature. 27 The side products are essentially a result of the zinc−ylide reaction with other present electrophiles (ester, aldehyde, and arylfluoride). Using the conditions developed for the synthesis of 13, ester 17 was readily transformed to acyl chloride 19.
Ethyl 5-chloromethyl-3,4-dichloro-1H-pyrrole-2-carboxylate 21 was prepared from commercially available 20 according to the literature procedure (Scheme 4). 18 After conversion to azide 22 by KI-mediated nucleophilic substitution, the reduction of 22 to amine 23 was first attempted via Pd/Ccatalyzed hydrogenation. This resulted in significant sideproduct formation, possibly via the nucleophilic attack of amine 23 to the electrophilic methylene moiety of 22, and aryl dehalogenation, as apparent from the 1 H NMR analysis of the crude reaction mixture. Avoiding the coexistence of amine and azide species during the reaction, we resorted to the milder Staudinger reduction, 28 which furnished amine 23 in 78% isolated yield. Saponification to 24, followed by phthalimide protection in neat phthalic anhydride gave 25 with 41% yield over four steps from 21.
To showcase the usefulness of the prepared building blocks in medicinal chemistry, the synthesis of compound 5, the analogue of antibacterial hit compound 1, was tackled (Scheme 5). After the smooth coupling of the acyl chloride, prepared from 25 in neat thionyl chloride, with the 2-aminobenzothiazole building block 29 in refluxing toluene, the deprotection step required some special attention. The formation of stable hydrazinium salt 27 was observed during the phthalimide deprotection, arguably due to the electronwithdrawing character of dichloropyrrole, which increases the acidity of the neighboring amides. It was crucial to first reprotonate the nitrogens of 27 to achieve complete deprotection after refluxing in ethanol overnight. Alkaline hydrolysis of phthalic hydrazide salt 28 yielded phthalate salt 29 and the anion was readily exchanged to the chloride salt of 5 by trituration with methanolic HCl.
Antibacterial hit compound 1 (c log P = 5.8) inhibited Escherichia coli DNA gyrase with IC 50 < 10 nM, and compound 5 (c log P = 2.0) inhibited the same enzyme with IC 50 < 10 nM. Moreover, 5 exhibits activity against Staphylococcus aureus (ATCC29213) with a minimal inhibitory concentration of 1 μg/mL. This confirms the hypothesis that the single-digit nanomolar inhibitory on-target activity coupled to the antibacterial activity can be retained while significantly reducing the lipophilicity by the modification of the pyrrole moiety.
To explore the reactivity and bioactivity of the fluorinated pyrroles, two additional analogues of 1 were prepared (Scheme 6) and evaluated for their on-target and antibacterial activities. Thus, compounds 31 and 33 inhibited E. coli DNA gyrase with IC 50 values of 32 and 150 nM, respectively, and possessed weak activity against S. aureus (ATCC29213) (31: MIC = 64 μg/mL; 33: MIC > 64 μg/mL).

CONCLUSIONS
In summary, practical synthetic routes to four new halogendoped pyrrole building blocks were developed, delivering the target compounds in sufficient quantities for further elaboration. Moreover, the transformation of the building blocks to potent DNA gyrase B inhibitors was demonstrated. Such building blocks are polar alternatives to molecular fragments found in naturally occurring or natural-product-inspired bioactive compounds and are useful in hit-to-lead optimization.